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Transformation Troubleshooting Guide
Transformation Troubleshooting Guide Resources » Technical Resource Centers » Gene Technical Resources » Molecular Cloning Center » Transformation Troubleshooting Guide Molecular Cloning Strategies What is PCR Restriction Enzymes Digestion DNA Ligation Plasmid
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Transformation Troubleshooting Guide
Resources » Technical Resource Centers » Gene Technical Resources » Molecular Cloning Center » Transformation Troubleshooting Guide
Molecular Cloning Strategies
What is PCR
Restriction Enzymes Digestion
DNA Ligation
Plasmid Transformation
DNA Transfection Guide
Transfection Protocol
DNA Transfection Troubleshooting
Ligation Protocol
DNA Ligation Troubleshooting Guide
PCR Protocol, PCR Steps
PCR Troubleshooting Guide
Restriction Digest Protocol
Restriction Digestion Troubleshooting Guide
DNA Ligation Troubleshooting
What is DNA Transformation|Biology
DNA Transformation Protocol
Transformation Troubleshooting Guide
A successful plasmid transformation is dependent on a number variables including antibiotic concentration, construct size and concentration, and ligation efficiency . Use the troubleshooting guide below to optimize your transformation reactions or get your desired gene in the vector you want the easy way with GenEZ™ ORF Clones. Start with a Search for your gene.
Molecular cloning strategies
PCR
Restriction digestion
Ligation
Transformation
Transfection
Molecular cloning handbook
Bioinformatics tools
Wrong antibiotic was used or antibiotic concentration was too high
Ensure the correct antibiotic was applied to plates.
Use only concentration recommended by competent cell or antibiotic manufacturer.
Competent cell viability is low
Thaw competent cells on ice and use immediately.
Check expiration date of cells.
Do not re-freeze cells.
Do not vortex cells - gently tap to mix.
Few or no colonytransformants
DNA insert encodes protein that is toxic to cells
Use a lower incubation temperature (25 – 30°C).
Use a cell strain and vector designed for tightly controlled transcription.
Heat-shock incubation too long
Reduce incubation time from 45 to 25 seconds.
Construct is too big
Use electroporation for vectors over 10 kb.
Too much ligation mixture was used for the transformation
Dilute ligation reaction with TE buffer (up to 5 times).
Too much DNA in reaction
Use no more than 1-10 ng of DNA in 5 µl for a 100 µl reaction or in 1-3 µl for a 50 µl reaction.
Low ligation efficiency
Vector insert ratio not optimal. Use a vector:insert molar ratio from 1:1 to 1:10.
Use a DNA concentration of 1-10 µg/ml.
Construct recombined with genomic DNA
Switch to a Rec A- cell strain.
No Plasmid inColony tranformants
Antibiotic concentration too low
Use antibiotic concentration recommended by manufacturer.
Antibiotic is degraded
Aliquot working volumes of antibiotic and avoid freeze-thaw cycles.
Add antibiotic to liquid plate media after sufficient cooling.
No insert in colonytranformants plasmids
Vector re-ligation
Vector insert ratio not optimal. Use a vector:insert molar ratio from 1:1 to 1:10. Use a DNA concentration of 1-10 µg/ml.
Dephosphorylate DNA with phosphatase to prevent re-ligation.
Sequencing of tranformantsplasmid reveals wrongplasmid sequence
Mutations introduced by initial PCR
Use a high-fidelity polymerase.
Inconclusive sequencing artifacts
Repeat sequencing reaction.
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